Tricarbonyl 99mTc(i) and Re(i)-thiosemicarbazone complexes: synthesis, characterization and biological evaluation for targeting bacterial infection

Article abstract of DOI:10.1039/c5dt02264a

Methyl, ethyl and phenyl nitrofuryl thiosemicarbazone ligands (1, 2 and 3 respectively) were radiolabeled with freshly prepared aqueous solution of a fac[^99mTc(CO)₃(H₂O)₃]⁺ precursor. The radiochemica

Full text of DOI:10.1039/c5dt02264a

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Tricarbonyl 99mTc(I) and Re(I)–thiosemicarbazone
complexes: synthesis, characterization and
biological evaluation for targeting
bacterial infection†
Cite this: DOI: 10.1039/c5dt02264a
Dipak Kumar Nayak,a Rinku Baishya,a Ramalingam Natarajan,b Tuhinadri Senc and
Mita Chatterjee Debnath*a
Methyl, ethyl and phenyl nitrofuryl thiosemicarbazone ligands (1, 2 and 3 respectively) were radiolabeled
with freshly prepared aqueous solution of a fac[99mTc(CO)3(H2O)3]+ precursor. The radiochemical yield
was around 98% as determined by thin layer chromatography and HPLC. The complexes exhibited sub-
stantial stability. The corresponding Re(I) complexes were prepared from a Re(CO)5Br precursor to under-
stand the coordination behavior of the ligands against a tricarbonyl rhenium(I) precursor. The rhenium(I)
complexes were characterized by means of IR, NMR and mass spectroscopic studies as well as by X-ray
crystallography, and correlated with the technetium complexes by means of HPLC studies. Electrochemi-
cal reduction of monomeric Re(CO)3-complexes of nitrofuryl ethyl thiosemicarbazone was also studied
using cyclic voltammetry. Biodistribution studies of 99mTc(CO)3-labeled thiosemicarbazones in rats intra-
muscularly infected with S. aureus exhibited substantial in vivo stability of the complex and moderate
accumulation at the site of focal infection.
Received 15th June 2015,
Accepted 4th August 2015
DOI: 10.1039/c5dt02264a
www.rsc.org/dalton
to the free thiosemicarbazones.9–11 Nitrofuryl thiosemicarb-
azones (NFT) have also been investigated as antibacterial and
Introduction
Over the last few years the chemistry of thiosemicarbazone antiprotozoal drugs.12,13 The electrochemical reduction of
complexes of transition metals has been receiving consider- nitro heterocyclic compounds follows a complex and well
able attention largely because of their wide range of biological known mechanism which is a prerequisite for their antibacter-
properties specially as antifungal, antibacterial, antiviral and ial activity.14,15 The highly reactive electrophilic intermediate
anticancer agents.1–4 Thiosemicarbazones with the general produced by the action of bacterial nitroreductases on NFT is
formula R1R2CvN–NH–CS–NR3R4 usually react as versatile postulated to affect bacterial DNA.3,16 Some pieces of evidence
chelating ligands and can coordinate with different transition suggest that the reduced NFT binds to bacterial ribosomes and
metal ions [e.g. Cu(II), Cr(III), Co(III), Ni(II) etc.] by bonding prevents protein synthesis.17 NFTs exert greater effects on bac-
through the sulfur and hydrazinic nitrogen atoms.5–8 The terial cells than on mammalian cells because bacterial cells
metal complexes show better biological activities as compared activate the drug more rapidly.18,19 Naturally, the preparation
of transition metal complexes of thiosemicarbazones contain-
ing the nitrofuryl pharmacophore and studies on their anti-
bacterial and antiparasitic activities are the areas receiving
much attention.12,13,20
aInfectious Disease and Immunology Division, Indian Institute of Chemical Biology
(CSIR), 4, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
E-mail: mita_chdebnath@yahoo.com, mitacd@iicb.res.in;
Technetium-99m is a transition metal and the most widely
used SPECT radionuclide in nuclear medicine that aids in the
diagnosis of various medical conditions.21–23 In the pursuit of
novel pharmacophores for use in infection diagnosis by scinti-
graphic imaging, we have already reported the labeling of
nitrofuryl thiosemicarbazones and a wide variety of fluoro-
quinolones with the fac[99mTc(CO)3(H2O)3]+ precursor.24 Con-
sidering the importance of nitrofuryl thiosemicarbazones, we
prepared 99mTc(CO)3 chelates of methyl, ethyl and phenyl
nitrofuryl thiosemicarbazone ligands (1, 2 and 3 respectively)
Fax: +91 33-2473-5197, +91 33-2472-3967; Tel: +033-2473 3491
bChemistry Division, Indian Institute of Chemical Biology (CSIR), 4,
Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India
cDepartment of Pharmaceutical Technology, Jadavpur University, Kolkata-700032,
India
†Electronic supplementary information (ESI) available: NMR, mass, IR spectra,
HPLC-figures, table of selected bond angles and CIF files giving X-ray crystallo-
graphic data. CCDC 878521, 1402565, 1017880, 1017882, 1017879 and 1402539
for Re(CO)3-1, trans-[ReBr(CO)3-1a], Re(CO)3-2, trans-[ReBr(CO)3-2a], cis-[ReBr-
(CO)3-2b] and Re(CO)3-3. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c5dt02264a
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of varying lipophilicities and investigated the influence of the
physicochemical behavior of the complexes on biological
activities in the present study.
Results and discussion
Synthesis and spectroscopic characterization
As a part of our efforts, we also tried to understand the Thiosemicarbazones 1–3 were synthesized in good yields
coordination behavior of nitrofuryl thiosemicarbazones (75–78%) by mixing equimolar amounts of nitrofurfural and
against the tricarbonyl rhenium(I) precursor. A series of thio- thiosemicarbazide derivatives in toluene (Scheme 1). A red
semicarbazone complexes were therefore prepared from colored amorphous powder was precipitated from the reaction
Re(CO)5Br, which proved to be an excellent starting material mixtures and was recrystallised from ethanol–water. The
for the synthesis of tricarbonyl rhenium(I) complexes. Thio- mother liquor was concentrated to recover more products. IR,
semicarbazones are versatile ligands that can coordinate NMR, mass spectroscopy and elemental analysis studies were
either as neutral ligands or in their deprotonated form. Both conducted to characterize the synthesized thiosemicarbazone
monomeric [ReBr(CO)3-LH] and dimeric {[Re(CO)3-L]2} com- derivatives.
plexes were produced and characterized by X-ray crystallo-
The pure thiosemicarbazone derivatives thus obtained were
graphic and spectroscopic studies. Electrochemical studies reacted with Re(CO)5Br in toluene to produce Re(CO)3–thio-
were carried out in order to determine the reduction potential semicarbazone complexes (Scheme 1) in good yields (74–78%).
of the nitroaromatic group. Thiosemicarbazones act in both HPLC analysis of the reaction mixture showed one sharp peak
types of complexes as bidentate N, S donors. 99mTc(CO)3 [RT = 13.9 min for {Re(CO)3-1}, 14.2 min for {Re(CO)3-2}, and
labeled thiosemicarbazones exhibited excellent stability in 16.1 min for {Re(CO)3-3}], attributed to the formation of mono-
in vitro and in vivo systems. Initial evaluations of the meric rhenium(I) complexes. Slow evaporation of the reaction
99mTc(CO)3-complexes in the S. aureus infected rat model were mixture from Re(CO)3-2 furnished two different crystals of
also performed.
monomers with different HPLC elution profiles [HPLC peaks
Scheme 1 Synthesis of ligands and complexes.
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Table 1 HPLC retention times for 99mtechnetium(I) and rhenium(I)
complexes
Compound
Retention time (min)
99mTc(I)-synthon
99mTc(CO)3-1
5.1
14.0
14.2
16.1
13.9
15.8
14.2
14.2
15.8
16.3
16.1
17.3
99mTc(CO)3-2
99mTc(CO)3-3
Re(CO)3-1 (monomeric)
[Re(CO)3-1]2
Re(CO)3-2 (monomeric)
trans-[ReBr(CO)3-2a]
cis-[ReBr(CO)3-2b]
[Re(CO)3-2]2
Re(CO)3-3 (monomeric)
[Re(CO)3-3]2
Fig. 1 Molecular structure of [Re(CO)3-1]2 with the atom-numbering
scheme, showing 30% probability displacement ellipsoids. Solvent mole-
cules have been omitted for the sake of clarity. Selected bond lengths
(Å): Re1–C1 1.928(7), Re1–C2 1.912(7), Re1–C3 1.894(7), C1–O1 1.136(7),
C2–O2 1.146(7), C3–O3 1.147(7), Re1–N1 2.174(5), Re1–S1 2.4446(16),
Re1–S2 2.5596(16), and S1–C4 1.763(5).
at 14.2 min for trans-ReBr(CO)3-2a and 15.8 min for cis-ReBr-
(CO)3-2b]. The HPLC retention times thus suggest that the
trans-monomer was initially formed, which got isomerised
during slow evaporation to produce the mixture of isomers.
Only one monomer (trans) could be isolated from the reaction
mixture for ReBr(CO)3-1 though evidence for the formation of
another, uncharacterized product could be obtained. On the
other hand, the concentration of the reaction mixtures led to
precipitates which could be crystallized. A HPLC study of the
products showed another sharp peak at later time points than
those mentioned above, attributed to the formation of dimeric
rhenium(I) complexes (Table 1).
The mass spectra of the monomeric complexes contain
signals corresponding to the molecular ion, although
[M − Br]+ formed the base peak. However with dimeric
Re(CO)3–thiosemicarbazone complexes, the [M/2 + H]+ species
resulting from the symmetric cleavage of the dimeric molecule
formed the base peak. A facial geometry around the rhenium
atom in all the complexes was suggested by two or three strong
IR bands (sometimes collapsed) for ν(CO) bond stretching in
the range 1909–2029 cm−1. The N–H group stretching bands
appeared at 3252–3400 cm−1. In the NMR spectrum, the
proton signals of furan hydrogen appeared deshielded (by
around 0.5 ppm) with respect to the free ligand. Other proton
signals remained nearly unchanged after co-ordination.
Fig. 2 Molecular structure of trans-(E)-ReBr(CO)3-1a with the atom-
numbering scheme, showing 30% probability displacement ellipsoids.
Solvent molecules have been omitted for the sake of clarity. Selected
bond lengths (Å): Re1–C1 1.891(7), Re1–C2 1.937(7), Re1–C3 1.934(7),
C1–O1 1.156(13), C2–O2 1.146(8), C3–O3 1.163(8), Re1–N1 2.226(5),
Re1–S1 2.4679(17), S1–C4 1.700(6), Re1–Br1 2.6688(8).
Crystallographic studies of Re(CO)3-complexes
As described above, the precipitate obtained by concentrating
the reaction mixture from the Re(CO)3-complex of ligand 1 was
dissolved in acetone containing few drops of ethanol, when
single crystals containing one molecule of acetone, [Re(CO)3-
1]2·C3H6O were formed. The complex was dimeric in nature,
and the molecular structure is shown in Fig. 1. The ligand
coordinates with the metal centre through the sulfur and azo-
methine nitrogen atoms. The rhenium atom is octahedrally co-
ordinated and interacts with two sulfur atoms, and the two
Re–S distances differ by about 0.1 Å, the values of Re1–S2
(S from neighboring monomer) and Re1–S1 (S from the same
monomer) being 2.559 and 2.444 Å respectively. These are in
accordance with the reported values of rhenium tricarbonyl
complexes of other thiosemicarbazone ligands.25,26 The de-
protonation of the ligand causes the replacement of the
halogen atom in the coordination sphere of the metal by the
sulfur atom of the neighboring monomer.
On the other hand, slow evaporation of the reaction
mixture (toluene) of the Re(CO)3 complex of ligand 1 furnished
crystals containing one molecule of toluene. X-ray crystallo-
graphic analysis revealed (Fig. 2) the formation of a mono-
meric complex, trans-[ReBr(CO)3-1a]. The dihedral angle
between C6–C5–N1–N2 is −170.5°. In this complex, the metal
forms a five membered chelate ring with the azomethine nitro-
gen and the sulfur atom of the thiosemicarbazone ligand.
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Fig. 4 Molecular structure of trans-(E)-ReBr(CO)3-2a with the atom-
numbering scheme, showing 30% probability displacement ellipsoids.
Solvent molecules have been omitted for the sake of clarity. Selected
bond lengths (Å): Re1–C1 1.892(11), Re1–C2 1.877(10), Re1–C3 1.930(13),
C1–O1 1.156(13), C2–O2 1.147(11), C3–O3 1.138(12), Re1–N1 2.215(6),
Re1–S 2.466(3), S–C4 1.690(9), and Re1–Br1 2.6627(10).
Fig. 3 Molecular structure of [Re(CO)3-2]2 with the atom-numbering
scheme, showing 30% probability displacement ellipsoids. Solvent mole-
cules have been omitted for the sake of clarity. Selected bond lengths
(Å): Re1A–C1A 1.929(13), Re1A–C2A 1.917(15), Re1A–C3A 1.905(16),
C1A–O1A 1.127(15), C2A–O2A 1.129(17), C3A–O3A 1.163(18), Re1A–N1A
2.187(9), Re1A–S1A 2.458(3), Re1A–S2A 2.552(3), and S1A–C4A 1.759(13).
Similarly, the concentration of the reaction mixture of the
Re(CO)3 complex of ligand 2 resulted in the precipitation of a
reddish brown solid that was stable in air, moderately soluble
in dichloromethane and highly soluble in acetone. Dark brown
single crystals were obtained by slow concentration of solution
of the precipitate in acetone and methanol. X-ray diffracto-
metry studies showed it to be the dimeric complex (cis isomer),
[Re(CO)3-2]2·C3H6O·CH3OH of ligand 2 (Fig. 3).
Two different types of single crystals were obtained by slow
evaporation of the reaction mixture (toluene) of the Re(CO)3
complex of ligand 2. Each molecule of the complex contains
one solvent molecule. The crystals differ in color, size and unit
cell parameters. The rhenium atom is octahedrally co-
ordinated to three carbonyl carbon atoms (in the fac arrange-
ment), a bromine atom, the azomethine nitrogen and the
sulfur atom of the thiosemicarbazone ligand, with which the
metal forms a five membered chelate ring. Structure elucida-
tion revealed the formation of two isomeric complexes trans-
[ReBr(CO)3-2a] and cis-[ReBr(CO)3-2b]. In the thiosemicarb-
azone backbone, the dihedral angle between C6–C5–N1–N2 is
−175.2° in the trans isomer (Fig. 4) but 4° in the cis isomer. No
remarkable change in bond lengths was observed for Re–N
and Re–S in the two isomers. Selected bond lengths are shown
Fig. 5 Molecular structure of [Re(CO)3-3]2 with the atom-numbering
scheme, showing 30% probability displacement ellipsoids. Solvent mole-
cules have been omitted for the sake of clarity. Selected bond lengths
(Å): Re1–C1 1.925(8), Re1–C2 1.916(10), Re1–C3 1.919(8), C1–O1
1.156(11), C2–O2 1.156(11), C3–O3 1.144(10), Re1–N3 2.182(7), Re1–S1
2.4594(19), Re1–S11 2.570(2), and S1–C4 1.778(7).
below the respective figures, which represent the molecular azomethine nitrogen atoms. As described above for 1 and 2,
structure of the two isomers (2a and 2b) studied by X-ray diffr- the thiosemicarbazone ligands were deprotonated in this case
action. Both the structures show the three carbonyl ligands also and the halogen atom in the coordination sphere of the
occupying one face and have a typical Re–C bond distance metal was displaced by the sulfur atom of the neighboring
(∼1.87–1.93 Å) of the facial Re(CO)3 unit.
ligand. Each Re is attached to two sulfur atoms in the dimers,
Finally, the concentration of an aqueous methanolic solu- one from the same monomer and the other belonging to the
tion of the initial precipitate of the Re(CO)3-complex of ligand neighboring monomer. There exist a difference of about 0.1 Å
3 furnished single crystals with one molecule of methanol, in the two Re–S distances. Re–N distances are comparable to
[Re(CO)3-3]2·CH3OH. The complex was dimeric in nature, the Re–N distances of the other two dimeric complexes. In the
whose molecular structure is shown in Fig. 5. The ligand case of dimeric and monomeric complexes, no remarkable
coordinates with the metal centre through the sulfur and difference in the Re–N distance was observed.
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Electrochemical studies
media.28 Reduction potentials of monomeric rhenium(I) com-
plexes were comparable to the values reported by other
workers in this field.
Fig. 6 shows typical voltammograms for monomeric Re(CO)3-
complexes of nitrofuryl ethyl thiosemicarbazone. Table 4 lists
the values of the first and second cathodic peaks. Voltam-
metric data indicated one electron reversible transfer (peak
Radiolabeling and quality control
IIIc/IIIa) around −0.80 V corresponding to the nitro-anion The 99mTc(I)-synthon was successfully prepared with more
radical generation while the more negative second peak could than 98% purity as verified by RP-HPLC (retention time
be attributed to the production of the hydroxylamine derivative 5.15 min). All three thiosemicarbazone ligands (1, 2 and 3)
(peak IVc).27 This study thus reveals the formation of the nitro were successfully labeled with the freshly prepared
anion radical as well as the hydroxylamine moiety. All of the
[
99mTc(CO)3(H2O)3]+ core. Radiolabeling yield depends on the
metal complexes exhibited lower E1/2 (E1/2 = (Ea + Ec)/2) values concentration of the ligand, pH of the synthon precursor and
than Nifurtimox (−0.88 V) as the reference showing a higher ligand solution, and the duration of heating. Complexation
capacity to be reduced, and hence a better ability to generate was favorable around neutral pH (7.0–7.5). Standardization
the radical species.
and optimization studies were performed to obtain maximum
Voltammograms of all the metal complexes show one complexation. Radiochemical purity of the complexes was
anodic peak (Ia) that could be attributed to the reoxidation routinely checked by thin layer chromatography (ITLC) on
process of the hydroxylamine RNHOH generated in IVc into silica gel plates and RP-HPLC. For all the complexes, the radio-
RNO. A second sweep exhibits a shoulder near IIc which could labeling efficiency was around 98% initially (30 min after
be attributed to the cathodic counterpart from the reversible preparation). It was around 92% even up to 24 h, indicating
reaction RNHOH ⇄ RNO + 2e− + 2H+. Prepeak IIc appears the stability of the labeled complexes. Radiochromatograms
even before the reduction of the nitro group, which seems (Fig. 7) showed a single peak eluting at 14.0, 14.2 and 16.1 min
to follow another reaction path beside the known electron- for 99mTc(CO)3-1, 99mTc(CO)3-2 and 99mTc(CO)3-3 respectively.
transfer mechanism of nitroaromatic compounds in aprotic The chemical identification of 99mTc-complexes was ascer-
tained by comparison of their HPLC profiles with those of the
corresponding rhenium(I) complexes (Table 1). The retention
times of 99mTc-complexes were comparable to those of the
corresponding monomeric Re-complexes. It is felt that techne-
tium-99m forms only monomeric complexes due to the high
dilution of the tracer.
In vitro stability
The in vitro stability of 99mTc(CO)3–thiosemicarbazone com-
plexes in normal saline was checked at various time intervals
for up to 24 h. For 99mTc(CO)3-1, 99mTc(CO)3-2 and 99mTc(CO)3-
3 the instant radiolabeling yields were 98.3 0.68%, 97.8
0.84% and 97.7 0.58% respectively. The percentage values of
the radiolabeled complex remaining after an incubation of 1 h
in saline were 98.2 1.09%, 97.3 1.63% and 97.3 0.97%
respectively, which changed to 93.1 2.74%, 91.9 1.74% and
92.0 1.34% after 24 h of incubation.
The in vitro serum stabilities of the three 99mTc(CO)3–thio-
semicarbazones were monitored by ITLC for up to 24 h at
37 °C. The levels of free radioactive species generated after
24 h of incubation were 8.74
2.51%, 9.64
3.07% and
9.23 2.77% for 99mTc(CO)3-1, 99mTc(CO)3-2 and 99mTc(CO)3-3
respectively.
Partition coefficient
The logarithm of partition coefficient (log Po/w) is a parameter
used to provide an indication of the lipophilicity of the com-
plexes. The log Po/w values as determined by partitioning
between octanol and phosphate buffer were found to be 0.52
0.06, 0.67 0.07, and 0.86 0.06 for 99mTc(CO)3-1, 99mTc(CO)3-2
and 99mTc(CO)3-3 respectively indicating the lipophilic nature
of the complexes.
Fig. 6 Cyclic voltammograms at 100 mV s−1 of 10 mM solutions of
(A) trans-[ReBr(CO)3-2a] and (B) cis-[ReBr(CO)3-2b] in DMF containing
0.1 M TBAP.
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Table 2 Crystal data and structure refinement parameters of Re(CO)3–thiosemicarbazone complexes
trans-(E)-ReBr(CO)3-
1a
trans-(E)-ReBr
(CO)3-2a
cis-(Z)-ReBr(CO)3-
2b
Compound
[Re(CO)3-1]2
C25H23N8O14Re2S2
1096.03
[Re(CO)3-2]2
C52H50N16O28Re4S4
2220.12
[Re(CO)3-3]2
C32H26N8S2Re2O14
1183.13
Empirical
formula
Molecular
weight
Crystal
system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
C13.5H11N4O6ReSBr
623.43
C18H18BrN4O6ReS
684.53
C18H18BrN4O6ReS
684.53
Triclinic
Triclinic
Monoclinic
Monoclinic
Monoclinic
Monoclinic
ˉ
P1
ˉ
P1
P21/n
P2(1)/c
P21
C2/c
7.5512(7)
10.6017(9)
12.8522(12)
94.519(5)
97.975(5)
90.201(4)
1015.66(16)
2
2.039
8.087
588.0
0.38 × 0.36 × 0.31
10.154(4)
10.283(4)
18.591(8)
94.160(9)
101.027(9)
110.970(8)
1757.9(12)
2
2.071
7.074
1046
0.22 × 0.17 × 0.12
11.1341(6)
18.3725(10)
11.6288(6)
90
101.055(3)
90
2334.7(2)
4
1.948
7.0954(6)
13.0042(10)
25.104(2)
90
97.113(3)
90
2298.5(3)
4
1.978
9.2120 (8)
41.834(4)
9.7296(9)
90
99.338(4)
90
3699.9(6)
2
1.993
24.149(5)
12.336(3)
13.934(3)
90°
111.929(3)
90°
3850.7(14)
4
2.041
γ (°)
V (Å3)
Z
Dc/g cm−3
μ/mm−1
F(000)
7.046
1312.0
0.23 × 0.21 × 0.19
7.157
1312.0
0.16 × 0.14 × 0.12
6.724
2124.0
0.16 × 0.14 × 0.10
6.467
2272.0
0.22 × 0.2 × 0.2
Crystal
dimensions
(mm)
Theta range
for data
collection
Limiting
indices
1.927 to 27.25°
2.15 to 25.00°
2.17 to 25.99
1.63 to 25.00°
1.95 to 27.84
1.81 to 25.99°
−9 ≤ h ≤ 9
−12 ≤ h ≤ 12
−11 ≤ k ≤ 12
−22 ≤ l ≤ 22
16 540/6163
−13 ≤ h ≤ 13
−21 ≤ k ≤ 22
−14 ≤ l ≤ 14
19 069/4570
−7 ≤ h ≤ 8
−11 ≤ h ≤ 12
−54 ≤ k ≤ 54
−12 ≤ l ≤ 12
35 570/ 16 427
[R(int) = 0.0392]
−29 ≤ h ≤ 29
−15 ≤ k ≤ 13
−12 ≤ l ≤ 17
9582/3775
−13 ≤ k ≤ 13
−16 ≤ l ≤ 16
18 391/4491
[R(int) = 0.0750]
−15 ≤ k ≤ 15
−29 ≤ l ≤ 29
16 489/ 3973
[R(int) = 0.0624]
Reflections
collected/
unique
[R(int) = 0.0376]
[R(int) = 0.0594]
[R(int) = 0.0549]
Refinement
method
Data/
Full-matrix least-
squares on F2
4491/260
Full-matrix least-
squares on F2
6163/464
Full-matrix least-
squares on F2
4570/315
Full-matrix least-
squares on F2
3973/ 270
Full-matrix least-
squares on F2
16 427/948
Full-matrix least-
squares on F2
3775/264
parameters
Goodness-of- 1.075
1.045
1.037
1.060
1.040
1.018
fit on F2
R indices [I > 2σ(I)]
2
R1 (on Fo
I > 2σ(I))
,
0.0439
0.0284
0.0556
0.0512
0.1181
0.0586
0.1743
0.0378
0.0855
0.0483
0.1378
2
wR2 (on Fo
I > 2σ(I))
,
0.1156
0.0440
0.0736
0.0737
0.0448
0.0566
wR2
0.1194
0.0602
0.1318
0.1940
0.0889
0.1528
Largest diff.
peaks and
holes
1.98 and
0.57 and
2.35 and
2.379 and
1.53 and
3.47 and
−1.78e A−3
−0.74e A−3
−1.61 e Å−3
−2.54 e Å−3
−1.19e Å−3
−1.80e Å−3
In vitro bacterial binding assay
carbazones. The above experiments were repeated with
99mTcO4 where three randomly selected bacterial samples
−
Table 5 represents the results of the bacterial binding assay
for 99mTc(CO)3-1, 99mTc(CO)3-2 and 99mTc(CO)3-3. Following
S. aureus incubation, 99mTc(CO)3-1 exhibited the highest
binding (8.14–8.56%) to the bacterial pellet followed by 99mTc-
(CO)3-2 (7.51–7.92%) and 99mTc(CO)3-3 (7.53–7.76%). The
above ranges are for three different radioactive concentrations.
No significant change in binding was observed after incu-
bation of the radiopharmaceuticals with the bacteria that had
been pre-exposed to a 50-fold excess of unlabeled ligands for
1 h, indicating non-specific binding. There was no significant
change in binding with increase or decrease in the drug level
in the radioactive dose. The viability of the cell suspension was
not affected after 2 h of incubation with 99mTc(CO)3–thiosemi-
were incubated with three aliquots of aqueous 99mTcO4
−
(activity range 185–525 kBq), showing significantly lower
binding (0.67–0.74%) to the above bacterial culture than that
observed with 99mTc(CO)3–thiosemicarbazones. No change in
the bacterial count was observed after 2 h of incubation. Also
no evidence of an antibacterial effect of radioactivity within
the stated range was found.
Biodistribution studies
The results of the biodistribution studies of the complexes in
rats exposed to S. aureus infection and having sterile inflam-
mation (turpentine) for different time periods are shown in
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Table 3 Selected bond angles (°) of Re(CO)3–thiosemicarbazones
trans-(E)-
ReBr(CO)3-
1a
trans-(E)-
ReBr(CO)3-
2a
cis-(Z)-
ReBr(CO)3-
2b
Angle
Angle
[Re(CO)3-1]2 Angle
Angle
Angle
[Re(CO)3-2]2 Angle
[Re(CO)3-3]2
C1–Re1–C3
C2–Re1–C1
C2–Re1–C3
C2–Re–Br1
C3–Re–Br1
C1–Re1–S1
C2–Re1–S1
C3–Re1–S1 173.1(2)
N1–Re1–S1
C1–Re1–N1
C2–Re1–N1 173.2(2)
C3–Re1–N1 96.9(9)
C1–Re1–Br1 179.7(2)
N1–Re1–Br1 83.36(14) S1–Re1–S2
C2–Re1–C1 86.0(3)
91.0(3)
86.0(3)
89.1(3)
93.7(2)
89.0(2)
C2–Re1–C1 90.0(3)
C3–Re1–C1 89.2(3)
C3–Re1–C2 89.6(3)
C1–Re1–C3
C2–Re1–C1
C2–Re1–C3
88.3(5)
86.5(6)
89.3(5)
C1–Re1–C2
C3–Re1–C2
C3–Re1–C1
C2–Re1–Br3 89.9(4)
C1–Re1–Br3 93.5(4)
C1–Re1–S1 173.8(5)
C2–Re1–S1
C3–Re1–S1
N3–Re1–S1
C1–Re1–N3
88.7(7)
89.5(6)
90.3(6)
C2A–Re1A–C1A 88.2(6)
C3A–Re1A–C2A 89.2(7)
C3A–Re1A–C1A 88.5(6)
C2–Re1–C1
86.9(12)
90.3(14)
88.5(13)
78.2(5)
C2–Re1–C3
C1–Re1–C3
N3–Re1–S1
N1–Re1–S1
N1–Re1–S2
78.66(12)
87.47(12)
95.62(19)
C2–Re–Br1 176.9(4)
N1A–Re1A–S1A
N1A–Re1A–S2A
78.0(2)
87.9(3)
C3–Re–Br1
C1–Re–S
C2–Re–S
C3–Re–S
N1–Re–S
89.1(4)
95.2(4)
94.5(4)
175.0(3)
78.9(2)
N3–Re1–S11 89.3(6)
95.15(19) C1–Re1–S1
94.5(2)
C1A–Re1A–S1A 175.8(4)
C2–Re1–S1
C1–Re1–S1
C3–Re1–S1 173.0(10)
C1–Re1–N3 175.2(10)
94.3(9)
97.0(8)
C2–Re1–S1 174.11(19)
C3–Re1–S1 92.28(19)
79.15(14) C1–Re1–N1 173.0(2)
97.3(4)
88.5(4)
79.6(3)
94.4(6)
C2A–Re1A–S1A
C3A–Re1A–S1A
95.1(4)
94.2(5)
C2A–Re1A–N1A 172.5(4)
C3A–Re1A–N1A 94.2(6)
C1A–Re1A–N1A 98.6(5)
C3A–Re1A–S2A 176.4(5)
96.9(2)
C2–Re1–N1 95.6(2)
C3–Re1–N1 94.9(2)
C1–Re1–N1 172.6(4)
C2–Re1–N3
C3–Re1–N3
C1–Re1–S1
92.8(10)
96.4(11)
97.0(8)
C2–Re1–N1
98.4(4)
97.3(4)
C2–Re1–N3 175.7(5)
C3–Re1–N3 93.4(5)
C3–Re1–Br3 176.2(4)
N1–Re1–Br1 84.39(18) N3–Re1–Br3 87.1(3)
C1–Re1–C3 88.3(5) C1–Re1–C2 88.7(7)
C1–Re1–S2
C2–Re1–S2
87.87(19)
95.31(19)
83.08(5)
C3–Re1–N1
C1–Re1–Br1 90.8(4)
C1A–Re1–S2A
S1A–Re1A–S2A
94.1(4)
83.32(9)
C1–Re1–S11 90.7(9)
S1–Re1–S11
C2–Re1–C1
81.7(2)
86.9(12)
C2–Re1–C1 90.0(3)
C2A–Re1A–C1A 88.2(6)
Scintigraphic imaging studies performed in the rat infec-
tion model revealed the potentiality of 99mTc(CO)3–thiosemi-
carbazones for the detection of bacterial infection. Retention
of the radiopharmaceuticals at S. aureus infected rat thigh
muscle at 4 and 8 h post injection time period is shown in
Fig. 8. The results were in accordance with the biodistribution
results in rats. The figures demonstrate the accumulation of
higher activity in the infected region as compared to the non-
infected tissues. They also reveal substantial accumulation of
the complexes in the liver and intestine suggesting that the
major route of excretion was hepatobiliary.
Thiosemicarbazones have been a subject of interest to
researchers of different disciplines due to their wide array of
pharmacological properties. Because of their potential anti-
bacterial and antiparasitic properties, studies of the co-ordination
chemistry of their transition metal complexes have been the
subject of interest in the biomedical field over the past few
years. Surprisingly, little is known about the technetium com-
plexes of thiosemicarbazones. Considering the importance
of technetium in the radiopharmaceutical field, namely,
in nuclear medicine, as well as the versatile application of
organometallic complexes in biological sciences we have radio-
labeled methyl, ethyl, and phenyl nitrofuryl thiosemicarb-
azones with the organometallic fac-[99mTc(CO)3(H2O)3]+
precursor and studied their antibacterial activities in in vitro
and in vivo systems. Chelation chemistry and the steric
profile are important considerations for the design of
kinetically stable small molecule imaging agents. Therefore,
detailed structural information was obtained by preparing
the corresponding rhenium(I) complexes as rhenium serves
as an effective non-radioactive chemical surrogate for
technetium.
Table 4 Cyclic voltammetric parameters for the reduction of Re(I) thio-
semicarbazone complexes measured in DMF at 100 mV s−1
Compound
EpIIIc (V) EpIIIa (V) ΔE (V) E1/2 (V) EpIVc (V)
trans [ReBr(CO)3-2a] −0.83
cis [ReBr(CO)3-2b] −0.80
−0.69
−0.65
0.14
0.15
−0.76
−0.73
−1.45
−1.40
Table 6. Following an intravenous injection, the drugs were
widely distributed in the animal body and excreted mainly
through the hepatobiliary route. For all the 99mTc labeled thio-
semicarbazones the uptake in the infected muscle was deter-
mined at 4, 8 and 24 h after injection. The percent injected
dose per g of bacteria infected or of the inflamed right thigh
muscle/control left thigh muscle was also determined in the
above three post injection time periods. The uptake of the
radiopharmaceuticals in the infected muscle was moderate at
the initial time periods (4 and 8 h) and gradually became less
up to 24 h. Infection vs. the normal tissue ratio was the
highest for 99mTc(CO)3-3 (4.62 : 1 at 4 h post injection). The
uptake in the inflamed muscle was also low. The blood pool
activity of the radiopharmaceuticals was considerably high and
clearance from the blood was very slow. This resulted in com-
paratively higher uptake levels in the liver, lung and spleen.
The compounds being lipophilic in nature are excreted mainly
through the hepatobiliary route. Localization of the radio-
activity in the liver and intestine was considerably high. Both
99mTc(CO)3-1 and 99mTc(CO)3-2 initially exhibited high
accumulation in the liver which gradually cleared out from the
organ. But 99mTc(CO)3-3 being most lipophilic exhibited very
poor clearance and a substantial amount of activity was
retained in the liver throughout the period of study. The
accumulation of radioactivity in the kidney was considerably
less (<1% ID per organ) resulting in poor renal excretion for all
groups. Accumulation of radioactivity in the stomach for all
the studied groups was significantly less, indicating the in vivo
stability of the labeled complexes.
In vitro stability studies of 99mTc(CO)3-1, 99mTc(CO)3-2 and
99mTc(CO)3-3 in saline and serum revealed minimal decompo-
sition of the label from the radiopharmaceuticals. Even after
an incubation period of 24 h, there was only 7–9% decrease in
the labeling efficiency, indicating the prolonged stability of the
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Fig. 7 (a) HPLC analysis profiles of 99mTc(CO)3-complexes of ligands 1, 2, and 3. (b) HPLC analysis profiles of Re(CO)3-complexes of ligands 1, 2,
and 3, as obtained from respective reaction mixtures and chromatograms of trans-[ReBr(CO)3-2a], and cis-[ReBr(CO)3-2b]. (c) HPLC chromatograms
of dimeric Re(CO)3 complexes of ligand 1 (A), ligand 2 (B), and ligand 3 (C).
99mTc labeled preparations and the suitability of the radio- pharmaceuticals accumulated at the in vivo sites of bacterial
labeled drugs for in vivo studies. The present study thus brings infection and exhibited an adequate target to non-target ratio.
out the suitability of 99mTc(CO)3–thiosemicarbazones for the However, their in vitro binding to microorganisms was not very
detection of focal infection in the rat thigh muscle. The obser- significant and this could not be blocked with an excess of
vations extend the scope of transition metal chelates of thio- unlabeled thiosemicarbazones.
semicarbazones for infection imaging/scintigraphy. The in vitro
The biodistribution studies gave evidence that the resulting
and in vivo study results have shown satisfactory radiolabeling complexes were stable in vivo during the study period and
of the thiosemicarbazone ligands with the 99mTc(CO)3(H2O)3 exhibited some accumulation in infection foci in the rat thigh
precursor, which was sufficiently stable up to 24 h and was model. These justify further studies with other thiosemicarb-
excreted mainly through the hepatobiliary routes. The radio- azone ligands, which are in progress.
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Table 5 In vitro binding assay reported as % radiotracer bound with S. aureus: (i) at three different doses either in the presence or absence of
excess unlabeled drug and (ii) 99mTc-pertechnetate
99mTc(CO)3-1
99mTc(CO)3-2
99mTc(CO)3-3
99mTc-pertechnetate
Compound
183 kBq 320 kBq 523 kBq
165 kBq 335 kBq 530 kBq
180 kBq 355 kBq 550 kBq
185
kBq
370
kBq
525
kBq
(40 μL)
(80 μL)
(120 μL)
(40 μL)
(80 μL)
(120 μL)
(40 μL)
(80 μL)
(120 μL)
Incubation
without excess
drug
Incubation
with excess
drug
8.14
0.14
8.56
0.15
8.34
0.20
7.92
0.13
7.51
0.17
7.64
0.22
7.76
0.15
7.62
0.24
7.53
0.26
0.67
0.07
0.74
0.09
0.69
0.02
8.23
0.21
8.37
0.14
8.46
0.15
7.84
0.11
7.56
0.18
7.69
0.20
7.67
0.15
7.70
0.03
7.39
0.06
NMR [δ (ppm), CD3OD]: 1.26 (t, 3H, CH3), 3.72 (q, 2H, CH2),
7.12 (d, 1H, furan H), 7.55 (d, 1H, furan H), 7.87 (s, 1H,
Experimental
All chemicals and solvents were either of analytical or HPLC –NvCH–). MS (ESI): m/z 243.1 [20%, (M + H)+], 265.1 [100%,
grade and used without further purification. 5-Nitrofurfural, (M + Na)+]. Anal. Calcd for C8H10N4O3S: C, 39.7; H, 4.2; N,
various thiosemicarbazides, p-TsOH, and bromopentacarbonyl 23.1; S, 13.2. Found: C, 39.4; H, 4.6; N, 22.7; S, 12.8.
rhenium(I) [Re(CO)5Br] were purchased from Sigma-Aldrich.
Data for nitrofuryl phenyl thiosemicarbazone (3). IR data
1
The other chemicals and solvents were procured from Merck (KBr, cm−1): ν 3342m, 3110m, 2965m (NH), 1542s (CvN). H
−
India. 99mTcO4 was obtained by 2-butanone extraction from NMR [δ (ppm), CD3OD]: 7.21 (d, 1H, furan H), 7.26 (m, 1H,
−
5 N NaOH solutions of 99MoO4 (purchased from BARC, phenyl H), 7.39 (t, 2H, phenyl H), 7.58 (d, 1H, furan H), 7.62
Mumbai, India). IR spectra were recorded on a Jasco 410 FTIR (m, 2H, phenyl H), 7.96 (s, 1H, –NvCH–). MS (ESI): m/z 313.1
spectrometer using KBr pellets. The 1H NMR spectra were [100%, (M + Na)+]. Anal. Calcd for C12H10N4O3S: C, 49.6; H,
recorded using a Bruker 300 DPX spectrometer. Mass spectra 3.5; N, 19.3; S, 11.0. Found: C, 49.4; H, 3.7; N, 19.1; S, 10.8.
were recorded using a Micromass Q-Tof (ESI-MS) spectrometer
or a Jeol JMS700 (FAB-MS) spectrometer. RP-HPLC studies
were performed using a Waters Associates HPLC system
99mTc-radiolabeling
Preparation of 99mTc(I)-synthon [99mTc(H2O)3(CO)3]+. The
equipped with a Waters 515 pump, and a 2489 dual wave-
length absorbance detector. Radioactivity in the RP-HPLC
eluates was monitored by using a Berthold LB-500 HERM
radio HPLC monitor procured from Berthold Technologies
GmbH, Germany. Various γ-countings were performed using a
well-type γ-counter from Electronic Corporation of India
(model LV4755). Cyclic voltammetry of monomeric Re(CO)3-
complexes was carried out using a PAR model 237A potentio-
stat/galvanostat.
tricarbonyl precursor was prepared as per the method reported
by us.24 Briefly, an aqueous solution (2 mL) of NaBH4 (11 mg),
Na2CO3 (8 mg) and Na/K tartrate (30 mg) taken in a sealed
glass vial was purged with CO gas (10–12 min). After the
addition of an aqueous solution of 99mTcO4 (1 mL, 200–250
−
MBq), the vial was heated at 75 °C for 20 min. After cooling
the vial and re-equilibrating with atmospheric pressure, the
pH of the reaction mixture was adjusted to 7 using a mixture
of 0.5 M phosphate buffer (pH 7.5) : 1 M HCl (1 : 3). The
synthon thus prepared was characterized by RP-HPLC.
General
Synthesis of ligands 1 to 3. Ligands 1 to 3 were synthesized
Labeling of ligands (1 to 3) with the [99mTc(H2O)3(CO)3]+
according to a previously published procedure.12 Briefly an precursor. Under optimized conditions, 0.8 mL of the freshly
equimolar mixture of 5-nitrofurfural and thiosemicarbazide prepared [99mTc(H2O)3(CO)3]+ precursor (120–150 MBq) was
was stirred in dry toluene at room temperature under a nitro- added to the ligand solution (2–2.5 mg dissolved in 0.2 mL of
gen atmosphere in the presence of p-TsOH (catalytic amounts) the EtOH–H2O mixture). The mixture was heated at 75 °C for
to produce the desired thiosemicarbazones in ∼75% yield 20 min and the pH was maintained between 7 and 7.5
(Scheme 1).
(Scheme 1). The purities of 99mTc(CO)3-complexes were ascer-
Data for nitrofuryl methyl thiosemicarbazone (1). IR data tained by TLC and RP-HPLC.
1
(KBr, cm−1): ν 3275m, 3210m, 3098m (NH), 1561s (CvN). H
General synthesis of the Re(CO)3-complexes of ligands (1 to
NMR [δ (ppm), CD3OD]: 3.16 (s, 3H, CH3), 7.11 (d, 1H, furan 3). To a solution of the ligand (0.25 mmol) in freshly
H), 7.55 (d, 1H, furan H), 7.87 (s, 1H, –NvCH–). MS (ESI): m/z distilled toluene (50 mL) was added an equimolar quantity of
229.0 [40%, (M + H)+], 251.0 [100%, (M + Na)+]. Anal. Calcd for Re(CO)5Br. The mixture was refluxed for 3 h, then concentrated
C7H8N4O3S: C, 36.8; H, 3.5; N, 24.5; S, 14.0. Found: C, 36.4; H, in vacuo to ca. 15 mL and maintained at room temperature for
3.6; N, 24.2; S, 13.8.
several hours (Scheme 1). A reddish brown solid that precipi-
Data for nitrofuryl ethyl thiosemicarbazone (2). IR data tated was filtered off, recrystallised from acetone and dried in
1
(KBr, cm−1): ν 3359m, 3104m, 2966m (NH), 1549s (CvN). H vacuum.
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Data for Re(CO)3-nitrofuryl methyl thiosemicarbazone [Re-
(CO)3-1]. Yield: 101 mg (81%). IR data (KBr, cm−1): ν 3348
1
(NH), 2027, 1909 (COfac). H NMR [δ (ppm), CD3OD]: 2.85 (s,
6H, CH3), 7.41 (m, 2H, furan H), 7.58 (m, 2H, furan H), 8.04 (s,
2H, –NvCH–). MS (ESI): m/z 498.9 [100%, (M/2 + H)+], 470.9
[35%, (M/2 − CO + H)+]. Anal. Calcd for C20H14N8O12S2Re2: C,
24.1; H, 1.6; N, 11.2; S, 6.4. Found: C, 23.8; H, 1.8; N, 10.9; S,
6.3.
The reddish brown solid was dissolved in acetone and few
drops of ethanol (C2H5OH) were added to obtain dark brown
single crystals of [Re(CO)3-1]2·C3H6O suitable for X-ray
diffraction.
Data for Re(CO)3-nitrofuryl ethyl thiosemicarbazone [Re-
(CO)3-2]. Yield: 121 mg (79%). IR data (KBr, cm−1): ν 3400
1
(NH), 2022, 1909 (COfac). H NMR [δ (ppm), acetone-d6]: 1.31
(t, 6H, CH3), 3.63 (br s, 4H, CH2), 7.59–7.64 (m, 4H, furan-H),
8.50 (br s, 2H, –NvCH–). MS (ESI): m/z 512.9 [100%, (M/2 +
H)+], 484.9 [20%, (M/2
− CO +
H)+]. Anal. Calcd for
C22H18N8O12S2Re2: C, 22.3; H, 1.7; N, 9.4; S, 5.4. Found: C,
22.0; H, 1.9; N, 9.2; S, 5.3.
The reddish brown solid precipitated upon the concen-
tration of the reaction mixture was redissolved in acetone and
few drops of methanol were added when dark brown single
crystals of the dimer [Re(CO)3-2]2·C3H6O·CH3OH suitable for
X-ray studies were furnished.
In a separate experiment, slow evaporation of the reaction
mixture (toluene) of the complex produced two different types
of single crystals (isomers 2a and 2b) of ReBr(CO)3–2·C7H8
suitable for X-ray diffraction.
Data for trans-[ReBr(CO)3-2a]. IR data (KBr, cm−1): ν 3350
1
(NH), 2029, 1926, 1902 (COfac). H NMR [δ (ppm), acetone-d6]:
1.32 (t, 3H, CH3), 3.68 (m, 2H, CH2), 7.78–7.83 (m, 2H, furan-
H), 8.67 (m, 1H, –NvCH–); additional peaks around 7.22 and
2.32 (ppm) are attributed to toluene used as the solvent for
crystallization. MS (ESI): m/z 513.1 [45%, (M − Br)+], 485.1
[100%, (M − Br − CO)+].
Data for cis-[ReBr(CO)3-2b]. IR data (KBr, cm−1): ν 3350
1
(NH); 2028, 1926, 1900 (COfac). H NMR [δ (ppm), acetone-d6]:
1.32 (t, 3H, CH3), 3.69 (m, 2H, CH2), 7.80–7.83 (m, 2H, furan-
H), 8.53 (m, 1H, –NvCH–); additional peaks around 7.22 and
2.32 (ppm) are attributed to toluene used as the solvent for
crystallization. MS (ESI): m/z 512.9 [75%, (M − Br)+], 484.9
[100%, (M − Br − CO)+].
Data for Re(CO)3-nitrofuryl phenyl thiosemicarbazone [Re-
(CO)3-3]. Yield: 124 mg (85%). IR data (KBr, cm−1): ν 3252
1
(NH), 2029, 1914 (COfac). H NMR [δ (ppm), acetone-d6]: 7.40
(m, 2H, furan-H), 7.55 (m, 8H, phenyl-H), 7.81 (m, 4H, phenyl-
2H, furan-2H), 8.62, 8.81 (s, 2H, –NvCH–). MS (ESI): m/z
1143.0 [85%, (M + Na)+], 583.0 [100%, (M/2 + Na)+], 561.0
[30%, (M/2 + H)+], 533.0 [30%, (M/2 − CO + H)+]. Anal. Calcd
for C30H20N8O12S2Re2: C, 32.1; H, 1.8; N, 10; S, 5.7. Found: C,
32.0; H, 2.1; N, 9.8; S, 5.6.
Red colored single crystals of [Re(CO)3-3]2·2CH3OH suitable
for X-ray diffraction were obtained by slow evaporation of a
methanolic solution of the reddish brown solid isolated from
the reaction mixture.
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X-Ray Solutions, USA) at 296 K. Suitable crystals were mounted
on a capillary using a Crystal Logic Synchrotron Goniometer
flat diffractometer that used graphite monochromated Mo Kα
radiation. The structures were solved by direct methods using
the program SHELXTL and refined by the full-matrix least
squares techniques on F2 using SHELXL-2013. The crystallo-
graphic data and structure refinement parameters are shown
in Table 2, while the selected bond angles and distances are
shown in Table 3 and below the respective figures respectively.
Quality control techniques
Thin layer chromatography. The radiochemical purities of
99mTc(CO)3-complexes of ligands 1–3 were determined by
instant thin layer chromatography (ITLC) on silica gel 60 F254
strips (1 × 8 cm, Merck KGaA, Darmstadt, Germany) spotted
with radioactive complexes and developed with methyl ethyl
ketone (MEK, system A) and brine (system B). The Rf values of
the 99mTc-complexes, 99mTcO4−, and [99mTc(H2O)3(CO)3]+ are
respectively 0, 1 and 0 in system A and 1, 1 and 0 in system
B. Quantitative analysis was performed by cutting the strips
into five pieces and counting them separately in a gamma
counter (ECIL, India).
Reverse-phase high-performance liquid chromatography
(RP-HPLC) analysis
The radiolabeling yields as well as characterization of the
[
99mTc(H2O)3(CO)3]+ precursor, and 99mTc(CO)3 and Re(CO)3-
complexes of ligands 1 to 3 were obtained by RP-HPLC analysis
using a Waters (USA) XTerra RP18 column (4.6 mm × 250 mm,
particle size, 5 μm) eluting with a gradient mixture of 0.1%
TFA in water (eluent A) and acetonitrile (eluent B). A linear gra-
dient was run at a flow rate of 1 mL min−1 for 0–25 min
(0–15 min: 90% A/10% B to 10% A/90% B and changing to
initial conditions between 15 and 25 min).
In vitro stability
The in vitro stability of the 99mTc(CO)3–thiosemicarbazone
complexes (1–3) was checked separately in normal saline and
freshly collected rat serum at different time intervals. A 0.1 mL
aliquot of each of the radiolabeled complexes was added separ-
ately to 0.9 mL of (a) saline and (b) serum, and incubated at
37 °C for 24 h. Samples were withdrawn from the mixture at 0,
0.5, 2, 4, and 24 h and analyzed by ITLC (silica gel plates/
methyl ethyl ketone developing solvent). The stability of the
radiolabeled complexes was also ascertained by His challenge
experiment. 0.1 mL aliquots of each of the 99mTc(CO)3-com-
plexes were added to 0.9 mL of 10−3 M histidine solution in
phosphate buffer (pH 7.4). The mixtures were incubated for
24 h at 37 °C and analyzed by ITLC (as above) at 1, 4 and 8 h.
Fig. 8 Scintigraphic images of 99mTc(CO)3–thiosemicarbazone in S.
aureus infected rats at 4 h and 8 h post-injection (i.v.): (I) 99mTc(CO)3-1,
(II) 99mTc(CO)3-2, (III) 99mTc(CO)3-3.
All the results are expressed as mean
experiments.
SD of three
Crystal structure analysis
Octanol water partition coefficient measurement
X-ray data collection, structure determination, and refine- The partition coefficient values (log Po/w) of the 99mTc(CO)3–
ment. Crystallographic data were collected on a Kappa APEX II thiosemicarbazone complexes (1–3) were determined by
Bruker SMART CCD area-detector system (Bruker Advanced adding 0.1 mL of each of the radiolabeled complexes to a cen-
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trifuge tube containing 1 mL 1-octanol and 1 mL phosphate into the right thigh muscle of each rat (body weight of 250 g).
buffer (0.025 M, pH 7.4). Each mixture was vortexed at room After 24 h the infection was apparent from swelling and red-
temperature for 2 min and then centrifuged at 3350g for dening of the inoculated muscle. Similarly sterile inflam-
5 min. From each phase equal aliquots (0.1 mL) of the organic mation was induced by injecting 0.1 mL of turpentine oil into
and aqueous layers were withdrawn and counted separately in the right thigh muscle of each rat belonging to a separate
an ECIL gamma counter. Each measurement was repeated group. Swelling appeared 24 h later. All the animal experi-
three times. Care was taken to avoid intermixing between the ments were approved by the Social Justice and Empowerment
phases. The partition coefficient (P) was calculated using the Committee for the purpose of control and supervision of
following equation: P = cpm in octanol − cpm in background/ experiments on animals (CPCSEA), Government of India, New
cpm in buffer − cpm in background and expressed as log Po/w
.
Delhi.
Cyclic voltammetry
Animal biodistribution studies
Cyclic voltammetry of the monomeric Re(CO)3 complex of
nitrofuryl ethyl thiosemicarbazone was carried out in DMF
(ca. 10 × 10−3 mol L−1), under a nitrogen atmosphere at room
temperature with TBAP (ca. 0.1 mol L−1) as the supporting
electrolyte. A Pt electrode (2 mm diameter) was used as a
working electrode and a Pt wire as a counter electrode. The
measurements were made against an Ag/AgCl reference elec-
99mTc(CO)3–thiosemicarbazone complexes (5–10 MBq per kg
body wt) were injected into the anaesthetized (ketamine
30–50 mg kg−1 IM) rats through a precannulated femoral vein.
The animals were sacrificed at 4, 8, or 24 h post injection.
Muscles from both infected and normal thighs and other
organs of interest were excised, rinsed with normal saline and
blotted dry to remove any residual blood. Blood samples and
urine were obtained by puncture of the heart and urinary
bladder respectively. Radioactivity of all samples was measured
using a well-type NaI(Tl) scintillation counter (ECIL, India)
against suitably diluted aliquots of the injected solution as
standards. The results are expressed either as percentage
injected dose per gram of tissue or percentage injected dose
per organ. Similar experiments were performed in the rat
model of turpentine induced sterile abscess. Abscess to
muscle activity ratios were calculated in both the infection and
sterile inflammation models.
trode with scan rates varying from 50 mV s−1 to 1000 mV s−1
.
In vitro bacterial binding study
Binding of 99mTc(CO)3–thiosemicarbazone complexes of
ligands 1–3 to a freshly prepared harvested culture of S. aureus
was assessed at 37 °C as per the reported method with some
modifications.29 Briefly, to each of three sterile Eppendorf
vials containing 1 mL of sterile PBS (0.1 mol L−1, pH 7.4) and
0.1 mL of the freshly prepared S. aureus culture (1 × 108 cfu
mL−1), one of the three different aliquots (40, 80 and 120 μL)
of 99mTc(CO)3–thiosemicarbazone complex solutions was
added. The mixtures were incubated for 1 h at 37 °C and then
centrifuged at 2000g for 5 min. The supernatants from each of
the three vials were transferred to three different test tubes.
The bacterial pellet was gently resuspended in sterile cold PBS
(1 mL), and recentrifuged as above; the supernatant was trans-
ferred to the respective previous vials containing the first
supernatant. The supernatants and pellets of each vial were
counted (ECIL γ-counter) separately against suitably diluted
aliquots of added radioactivity (to each vial) as the standard.
For each concentration of each of the radiolabeled thiosemi-
carbazones, the experiments were repeated four times and the
results are expressed as the mean S.D. The above procedure
Conclusion
In summary three nitrofuryl thiosemicarbazone ligands were
successfully radiolabeled with the fac-[99mTc(H2O)3(CO)3]+
core. The in vitro results have shown satisfactory radiolabeling
of the ligands with tricarbonyl technetium. The in vivo behav-
ior of the complexes was evaluated by biodistribution studies
in the infected rat model. The reactions of ReBr(CO)5 with
different nitrofuryl thiosemicarbazone ligands yielded the
corresponding Re(CO)3–thiosemicarbazone complexes, which
were characterized by X-ray crystallography. The structure elu-
cidation helped us in understanding the coordination chem-
istry of thiosemicarbazone ligands with the technetium metal
which will be useful in designing new target specific 99mTc
and 186/188Re–thiosemicarbazone radiopharmaceuticals based
on the tricarbonyl concept. Electrochemical behaviors of
monomeric complexes mainly focus on the reduction of the
nitro group, which is a prerequisite for antibacterial effects. If
suitably developed, these compounds can also be used for
hypoxia imaging.
was also repeated with 99mTcO4 (185, 370 and 525 kBq) in
−
separate experiments. The experiments for each concentration
of each of the radiolabeled complexes were also repeated sim-
ultaneously in the presence of 50-fold excess of unlabeled
ligands to determine the specificity of 99mTc(CO)3-labeled thio-
semicarbazone binding to bacteria. The bacterial culture was
preincubated for 1 h with excess unlabeled thiosemicarb-
azones and all other processes were similar as before.
Induction of infectious foci and non-infected inflammation
in rats
Statistical analysis
A freshly prepared harvested culture of S. aureus (1 × 108 cfu
mL−1) in 0.1 M PBS (pH 7.4) was used to produce a focal infec- All mean values of animal experiments are expressed as % ID
tion and 0.2 mL of the above bacterial suspension was injected per g of tissues or organs SD. The p values of the experi-
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mental results were calculated and found to be <0.05 (statisti-
cally significant).
C. Olea-Azar and M. Faundez, Bioorg. Med. Chem., 2004, 12,
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13 S. Sharma, F. Athar, M. R. Maurya, F. Naqvi and A. Azam,
Eur. J. Med. Chem., 2005, 40, 557–562.
Conflict of interest
14 M. M. B. Pessoa, G. F. S. Andrade, M. R. Santos and
M. L. A. Temperini, J. Electroanal. Chem., 2003, 545, 117–
122.
The authors declare no competing financial interest.
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16 L. Otero, J. D. Maya, A. Morello, C. Rigol, G. Barriga,
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18 C. Bot, B. Hall and G. Alvarez, Antimicrob. Agents Chemo-
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Acknowledgements
This work was funded by the Department of Atomic Energy,
Mumbai (India) and by the Council of Scientific and Industrial
Research (India), we thank both organisations for financial
support. We thank Dr Basudeb Achari, Emeritus Scientist
for his valuable suggestions during the preparation of the
manuscript.
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